Reidentification of Sex Pheromones of Tea Geometrid Ectropis Obliqua Prout (Lepidoptera: Geometridae)

Journal of Economic Entomology, 109(1), 2016, 167–175 doi: 10.1093/jee/tov282 Advance Access Publication Date: 21 October 2015 Ecology and Behavior Research article

Reidentification of Sex Pheromones of Tea Geometrid Ectropis obliqua Prout (Lepidoptera: Geometridae)

Yunqiu Yang,1 Longwa Zhang,2 Feng Guo,1,3 Yanhua Long,3 Yun Wang,1 and Xiaochun Wan1,4

1State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, China ([email protected]; [email protected]; [email protected]), 2Provincial Key Laboratory of Microbial Pest Control, Anhui Agricultural University, Hefei 230036, China ([email protected]), 3School of Life Science, Anhui Agricultural University, Hefei 230036, China (yyq_ly- [email protected]), and 4Corresponding author, e-mail: [email protected].

Received 26 March 2015; Accepted 2 September 2015 Downloaded from Abstract Tea geometrid Ectropis obliqua Prout (Lepidoptera: Geometridae) is an important defoliator of the tree crop Camellia sinensis L. in China. The sex pheromones of E. obliqua have not been identified, but have potential importance relative to the biological control of this predator. In this study, the female sex pheromones of E. obliqua were identified and evaluated for use in the monitoring and mass trapping of this pest. The sex pheromone http://jee.oxfordjournals.org/ extracts were subjected to gas chromatography–electroantennographic detection and gas chromatography– mass spectrometry. The identified chemicals were synthesized and applied to wind-tunnel tests and field experiments. (Z,Z,Z )-3,6,9-octadecatriene and 6,7-epoxy-(Z,Z )-3,9-octadecadiene were determined to be the primary sex pheromones produced by the female E. obliqua; the latter elicits the strongest electroantennogram responses from male E. obliqua antennae. However, males did not respond to single components in the wind- tunnel tests. The results of a field-trapping experiment indicated that a 4:6 v/v blend of (Z,Z,Z )-3,6,9-octadecatriene and 6,7-epoxy-(Z,Z )-3,9-octadecadiene was highly effective in attracting male moths.

Key words Ectropis oblique Prout, sex pheromone, (Z,Z,Z )-3,6,9-octadecatriene, 6,7-epoxy-(Z,Z )-3,9-octadecadiene, Camellia by guest on March 21, 2016 sinensis L

Tea geometrid Ectropis obliqua Prout (Lepidoptera: Geometridae 9-nonadecatriene as sex pheromones of E. obliqua (Li et al. 1988). Ennominae) is a major pest that infests thousands of hectares of tea Yao et al. (1991) deduced that (Z,Z,Z )-3,6,9-octadecatriene and plantations (Camellia sinensis L) in China annually. E. obliqua can 6,7-epoxy-(Z,Z )-3,9-octadecadiene were sex pheromones of this also be found in Japan, but it is not considered a serious pest in that pest through gas chromatography–mass spectrometry (GC-MS). country (Hu et al. 1994). Outbreak populations of this pest can com- These authors also reported that (Z,Z,Z )-3,6,9-docosatriene and pletely consume all the leaves on a tea bush, thereby limiting tea pro- (Z,Z,Z )-3,6,9-tetracosatriene exhibit a certain capability to duction in summer and autumn severely and affecting tea production attract(Yao et al. 1991). However, Yao et al. did not present the for the following year (Hazarika et al. 2009, Zhang et al. 2014). E. field attraction data and the statistical analyses or details about the obliqua is multivoltine and produces six to seven generations per year doses and dispensers used. (Yang et al. 2008). Recurring E. obliqua outbreaks can be controlled The sex pheromones of E. obliqua have not been accurately iden- with large doses of chemical pesticides; however, this practice leaves tified; therefore, the use of pheromones to monitor and control this pesticide residues on the tea that is shipped to the market and pollutes moth in tea orchards has been hampered. In the present work, we the environment (Hazarika et al. 2001, Ye et al. 2014). As such, non- examine the active compounds extracted from the female sex phero- insecticidal measures must urgently be developed to control E. obli- mone glands of E. obliqua, the sensory responses of male moths to qua; the utilization of sex pheromones to manipulate behavior is a synthetic compounds, and the behavioral responses of male moths promising alternative to pesticides (Hazarika et al. 2009). as per wind-tunnel and field bioassays. Attempts to identify the sex pheromones of E. obliqua were initi- ated more than 30 yr ago. Du (1978–1981) studied E. obliqua sex Materials and Methods pheromones and identified multiple potential components, but the chemical structures of these components were not reported (date Insects unpublished). Li et al. (1988) identified (Z,Z,Z )-3,6,9-octadeca- E. obliqua insects were obtained from Qian-Shan County (31.5 N, trienyl acetate, (Z,Z,Z )-3,6,9-octadecatriene, and (Z,Z,Z )-3,6, 116.3 E), Anhui Province, China. Approximately 10 generations of

VC The Authors 2015. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: [email protected] 167 168 Journal of Economic Entomology, 2016, Vol. 109, No. 1 this moth were maintained in the laboratory. Fresh material was col- although we modified their process slightly (Fig. 1). We used methyl lected regularly from the field and introduced into the laboratory linolenate (“1”) instead of linolenic acid to react with LiAlH4 in culture to prevent degeneration, and the larvae were reared on tea THF to produce the corresponding 9,12,15-octadecatrien-1-ol leaves. Furthermore, both adults and larvae were maintained under (“2”). Alcohol reacted with 4-methylbenzene-1-sulfonyl chloride controlled conditions at 22 6 3C, 60–70% RH, and a photoperiod (TosCl) in DCM converted to tosylate (“3”); then, tosylate reacted of 14:10 (L:D) h. Scotophase and photophase were reversed from with LiAlH4 in ether. Once the solvent evaporated, the crude prod- the natural light cycle to permit scotophase observation during nor- uct was purified through chromatography (PE ¼ 100%) to obtain mal working hours. The pupae were sexed based on the morphology (Z,Z,Z )-3,6,9-octadecatriene (“4”). The purity of the produced of the eighth-tenth abdominal segments and were maintained in (Z,Z,Z )-3,6,9-octadecatriene was >98%. (Z,Z,Z )-3,6,9-octadeca- moist sand for eclosion. The adults were housed separately in 240- triene MS (EI. 70 eV): 248 (1) [Mþ], 192 (11), 135 (6), 108 (41), 95 ml plastic jars and fed with 10% honey solution soaked in cotton. (32) [C7H11þ], 67 (60) [C5H7þ], 79 (100) [C6H7þ], 55 (44) [C4H7þ], 41 (70). Female Calling (Z,Z,Z)-3,6,9-octadecatriene was converted to a mixture of the The diel rhythm of calling behavior was observed in 1- to 3-d-old three corresponding mono-epoxydienes through oxidation with m- virgin females (N ¼ 50) that were individually placed in plastic con- chloroperoxybenzoic acid in dichloromethane (Ando et al. 1993). tainers and housed under the artificial environment. Observations The crude product was separated with a reversed-phase column were conducted under red light (3 lux) at 1-h intervals during (SEPAX-GP18, Sepax Technologies, Inc., Newark, DE) and eluted scotophase. with 80% acetonitrile. A portion of the three regio-isomers was then separated into individual monoepoxides with silica gel (200–300

mesh; Sinopharm Chemical Reagent Beijing Co., Ltd.) and then Downloaded from Extraction of Sex Pheromones eluted with 50% benzene in hexane. The elution order was 6,7- Active sex pheromones were extracted from the glands of 1-day-old epoxide < 9,10-epoxide < 3,4-epoxide. The purity of the produced virgin females (N ¼ 20) 5 h after the scotophase began. Mating was 6,7-epoxy-(Z, Z)-3,9-octadecadiene was 95% with respect to the most frequently observed during this period. The terminal abdomi- other positional isomers. 6,7-epoxy-(Z,Z)-3,9-octadecadiene MS nal tip, including the pheromone gland, was excised from the virgin (EI. 70 eV): 264(1) [Mþ], 235(1) [C16H27Oþ], 181 (2), 195 (1) female moth. The tip was immersed in 10 ll of redistilled hexane for http://jee.oxfordjournals.org/ [C13H23Oþ], 111(13) [C7H11Oþ], 97 (18), 95 (25), 81 (40), 79 4–6 h at room temperature. Experimental procedures were per- (27), 67 (100), 55 (78), 41 (82). formed under red light (3 lux) to facilitate observation and to avoid disturbing the insects. Then, the tip was removed and the extracts merged for either gas chromatography–electroantennogram (GC- Electrophysiology EAG) or GC-MS analyses without purification. EAG was conducted with an EAG apparatus (Syntech Co., the Netherlands) to test the electrophysiological activity of male E. obli- GC-EAG and GC-MS Analyses qua antennae in response to different doses of (Z, Z, Z)-3,6,9-octadecatriene and 6,7-epoxy-(Z, Z )-3,9-octadecadiene. These antennae

The EAG activity of the natural E. obliqua pheromone components by guest on March 21, 2016 was determined using a gas chromatograph equipped with an elec- were prepared for GC-EAD by exposure to 4 ml/s of humidified and troantennographic detector (EAD; Struble and Arn 1984). An charcoal-filtered air through a 35-cm-long glass tube (inner diame- Agilent 7890 gas chromatograph was equipped with an INNOWAX ter, 8 mm; outer diameter, 10 mm). Approximately 3-mm holes were capillary column (60 m by 0.35 mm i.d. by 0.25 lm, J&W Scientific bored into the glass tube to facilitate the insertion of a Pasteur pip- Palo Alto, CA, USA). The column temperature was programmed at ette and to administer the pheromone test stimuli. The test stimuli 50C for 2 min and increased at a rate 5C/min to 250C, with a used were serial hexane dilutions (0.001, 0.01, 0.1, and 1 mg/ml) of final hold of 5 min. The effluent from the column was split into two the compounds. These stimuli (10 ll) were applied to a 1-cm by 5- lines that were connected to a flame ionization detector and to EAD cm piece of filter paper, which was placed in the Pasteur pipette at a ratio of 1:1. Antennae were excised from either a male or female once the solvent (hexane) had been left to evaporate for 5 min. The moth at the base, and a few distal segments were cut to enhance con- test stimuli were then delivered in 0.5-s pulses of 4 ml/s of air by a tact with the saline in the electrodes. Each end of the antenna was stimulus controller (type CS-55) to transport volatiles to the antenna connected to two recording electrodes with Spectra 360 electrode for stimulation. The EAG signal was amplified (10) with an AC/ gel (Parker Laboratories Inc., Orange, NJ) to ensure that the sensilla DC UN-6 amplifier in DC mode (Syntech Laboratories, Hilversum, faced the efﬂuent ﬂow from the GC. Each EAD analysis was repli- The Netherlands). A minimum period of 30 s was set between cated five times. administrations of test stimuli to recover antennal responsiveness. An HP-5793 mass spectrometer in electron impact (EI) mode Redistilled hexane (10 ll) was used as a control stimulus in every (70 eV) interfaced with an HP-6890N gas chromatograph was used test. The absolute EAG amplitudes (mV) obtained following the sub- to obtain mass spectra using the same columns (INNOWAX) and traction of the solvent response were used for data analysis. Each temperature program as in the GC-EAD analysis. The ion source, stimulus was tested on the antennae of 10 different males, and the injector, and detector temperatures were all set to 250C. Helium obtained data were analyzed by one-way ANOVA, followed by a was used as a carrier gas. least significant difference (LSD) multiple comparison test at GC-EAD active compounds were identified by observing the P < 0.05 (SPSS 11.0 for Windows, 2002; SPSS Inc., Chicago, IL). characteristic ions and by comparing their spectra and retention times with those of authentic compounds. Wind-Tunnel Bioassays A glass wind tunnel (300 by 50 by 50 cm) was constructed to test Chemical Synthesis the biological activity of male E. obliqua in response to binary (Z,Z,Z )-3,6,9-octadecatriene and 6,7-epoxy-(Z,Z )-3,9-octadeca- blends of (Z,Z,Z )-3,6,9-octadecatriene and 6,7-epoxy-(Z,Z )-3,9- diene were synthesized as described by Liu (Liu et al. 1994), octadecadiene (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9, and Journal of Economic Entomology, 2016, Vol. 109, No. 1 169 Downloaded from http://jee.oxfordjournals.org/ by guest on March 21, 2016 Fig. 1. Synthesis of authentic standards.

0:10). The activity of different lures was compared with that of one Field Experiments equivalent female (FE) gland extract. Field-trapping experiments were performed in Qian-Shan County in Bioassays were performed under red light (3 lux) at 25 6 2C, June 2014 to verify the attractiveness of the pheromone components 60% 6 5% RH, and air speeds of 40–50 cm/s. Air flow was gener- to male E. obliqua and to develop an optimized lure trap. Rubber ated and purified (activated charcoal) with a centrifugal fan and septa (6 mm OD, Pherobio Technology Co. Ltd, Beijing, China) conducted through two metallic net (3-mm mesh) screens to achieve were used as pheromone dispensers. Each septum was placed at the laminar flow. Prior to each experiment, 10 ll of hexane solution center of a sticky trap (30 by 27 cm) with sticky inserts to capture (1 lg/ll) that consists of each lure to be tested was distributed on a moths. The traps were set at a height of 1.3 m above the ground, circular piece of the filter paper (1.0 cm2). After solvent evaporation and spaced 10 m apart. The trap positions within each replicate (3 min), the lure was suspended on a stainless steel holder at a dis- were re-randomized, and the sticky inserts were replaced daily after tance of 25 cm from the upwind end of the tunnel in the middle of the traps were checked. In the experiment, the attraction of male E. the cross section. Experiments were conducted with 1 - to 2-d-old obliqua to various combinations of the two candidate compounds virgin males that were individually released from a cylindrical (4 cm was tested. The treatments were as follows: blank control, 10:0, 8:2, i.d. by 6 cm) metallic net (3 by 3 mm mesh) cage suspended approxi- 6:4, 5:5, 4:6, 2:8, and 0:10 lg loadings of (Z,Z,Z )-3,6,9-octadeca- mately 15 cm from the top and positioned 150 cm from the source. triene:6,7-epoxy-(Z,Z )-3,9-octadecadiene (N ¼ 8). Data were sub- These male moths were given 10 min to respond. The behavioral jected to ANOVA, followed by the LSD test (P < 0.05). sequences recorded were as follows: wing fanning (WF), taking flight (TF), orientation toward the odor source (OR), and source contact (SC). If the male remained still for 10 min in the wind tun- Results nel, this behavior was regarded as a lack of response. The male Diel Rhythm of Female Calling insects (20 per treatment) were used once. For each behavioral cate- To determine the age and extraction time of pheromone glands, gory, the percentages of responding males were subjected to mating behavior was observed in the laboratory. Most of the ANOVA, followed by Ryan’s multiple comparison test on propor- females exhibited calling behavior (extrusion of the last urite) during tions (P < 0.05). the first (87.5%) and second (92.5%) scotophases following emer- The interval between administrations of blends was at least 1 h gence. Some females continued calling during the third scotophases after the tunnel was cleaned with hexane and allowed to dry. (42.5%), whereas others died on the third day. 170 Journal of Economic Entomology, 2016, Vol. 109, No. 1

During scotophase, females started their calling behavior from between C8 and C9 (with hydrogen transfer) and exhibited the char- the first hour of darkness; the calling rate peaked at the fifth hour acteristic of methylene-interrupted polyenes with one outer double and then continued until the beginning of photophase (Fig. 2). Based bond at C3. Another distinct ion at M-56 (m/z ¼ 192; loss of C4H8) þ on these observations, extracts were obtained from 1-d-old females is [H(CH¼CH)3R] , which is attributed to the rearrangement approximately 5 h after scotophase began. (Millar et al. 1987, Ando et al. 2004) and cleavage between C4 and

C5; this ion places the other outer double bond between C9 and C10 (De Silva et al. 2013). Identification of Pheromone Components Compound II was identified as 6,7-epoxy-(Z,Z )-3,9-octadeca- Figure 3 shows the results of the GC-EAD analysis of the crude diene, with diagnostic ions at m/z 97, 235 (M-29), 195 (M-69), 111, extract of the sex glands on an INNOWAX capillary column. The and 264 (Mþ)(Fig. 4II). Although diagnostic fragmentation with pheromone extracted from E. obliqua females revealed two com- relatively intense signals was observed in the low-mass region, a pounds at retention times of 28.2 (I) and 34.9 min (II). The strongest small but diagnostic fragment at m/z 97 (C H O)þ strongly sup- responses were elicited by compound II, which consistently stimu- 6 10 ported the presence of 6,7-epoxide. By contrast, the distinctive frag- lated responses from the antennae of male moths (Fig. 3A). ment (M-69 ¼ m/z 195) from the a cleavage to the epoxide However, the antennae of female moths were not stimulated by the supported the structural assignment of 6,7-epoxy-3,9-octadeca- pheromone extract (Fig. 3B). The difference in the EAG responses of diene. Assuming the existence of close biogenic relations between the males and females reflected the differences in the structure, func- the two active compounds, the structure of the second component tion, and behavior of their olfactory systems (Wang et al. 2009). was more likely to be that of an epoxydiene than that of a ketodiene In the GC-MS analyses, the mass spectrum of compound I was because no characteristic acylium ion or McLafferty rearrangement identified as (Z,Z,Z )-3,6,9-octadecatriene, with diagnostic fragment

product was detected (Hansson et al. 1990; Ando et al. 1993, 1995). Downloaded from ions at m/z 67,79 (base peak, [H(CH¼CH) ]þ), 108,192,248 (Mþ) 3 Among the three monoepoxydienes derived from (Z,Z,Z )-3,6,9- (Fig. 4I). The large diagnostic peak at m/z 108 [CH CH 3 2 octadecatriene, 3,4-epoxy-(Z,Z )-6,9-octadecadiene (Fig. 5A) and (CH¼CH) H]þ originated from the structure-specific cleavage 3 9,10-epoxy-(Z,Z )-3,6-octadecadiene (Fig. 5C) exhibited mass spectra that differed from those of the natural compound. A comparison of the retention times of the compounds (I and II) with those of authentic samples (Z,Z,Z-3,6,9-octadecatriene and http://jee.oxfordjournals.org/ 6,7-epoxy-3,9-octadecadiene) confirmed the identities of these compounds (Fig. 6).

Electroantennography Figure 7 depicts the male EAG response to different doses of the sex pheromone compounds. The responses of the EAGs to any dose of

6,7-epoxy-(Z,Z )-3,9-octadecadiene were significantly higher than by guest on March 21, 2016 Fig. 2. Diel rhythm of the calling behavior of E. obliqua adult under artiﬁcial the responses to (Z,Z,Z )-3,6,9-octadecatriene (P < 0.05). No signifi- photoperiod. cant differences were observed between 1 and 10 lg doses of the

Fig. 3. Simultaneously recorded gas chromatography–electroantennographic detection for E. obliqua with sex gland extracts. Journal of Economic Entomology, 2016, Vol. 109, No. 1 171 compounds. 6,7-epoxy-(Z,Z )-3,9-octadecadiene elicited the highest orientation toward SC. This response was similar to that induced by EAG (3 mv) responses from male antennae at a dose of 1 lg. the 10EF gland extract and was significantly superior to those evoked by the other mixtures (P < 0.05). Wind-Tunnel Bioassay None of the males responded to single components in the single- Field Trapping component test (Table 1). In the binary blend test, the 4:6 blend of In the field-trapping experiment (Table 2), a significant difference (Z,Z,Z )-3,6,9-octadecatriene and 6,7-epoxy-(Z,Z )-3,9-octadeca- was observed in the number of male moths captured through differ- diene elicited 100% male orientation toward OR and 90% male ent treatments. Traps baited with a 4:6 ratio of (Z,Z,Z )-3,6, Downloaded from http://jee.oxfordjournals.org/ by guest on March 21, 2016

Fig. 4. Mass chromatogram and diagnostic fragment of compounds I and II. 172 Journal of Economic Entomology, 2016, Vol. 109, No. 1 Downloaded from http://jee.oxfordjournals.org/ by guest on March 21, 2016

Fig. 5. Electron impact mass spectra (70 eV) of 3,4-epoxy-(Z,Z)-6,9-octadecadiene (A), 6,7-epoxy-(Z,Z)-3,9-octadecadiene (B), and 9,10-epoxy-(Z,Z)-3,6-octadecadiene (C). Journal of Economic Entomology, 2016, Vol. 109, No. 1 173 Downloaded from http://jee.oxfordjournals.org/ Fig. 6. Total ion chromatogram of GC-MS analysis of the gland extracts of female (A) and chemical standards (B). I: standard compound (Z,Z,Z)-3,6,9-octadecatriene; II: standard compound 6,7-epoxy-(Z,Z)-3,9-octadecadiene; internal standards: ethyl heptanoate.

The GC-EAD and GC-MS results highlighted two active compounds, namely, (Z,Z,Z )-3,6,9-octadecatriene and 6,7-epoxy- (Z,Z )-3,9-octadecadiene; no other compound in the crude extract of the female sex gland elicited responses. (Z,Z,Z )-3,6,9-octadeca-

triene and 6,7-epoxy-(Z,Z )-3,9-octadecadiene are either major or by guest on March 21, 2016 minor components of sex pheromones in other lepidoptera species, particularly those in the families Noctuidae and Geometridae. For instance, (Z,Z,Z )-3,6,9-octadecatriene is a component of Semiothisa signaria dispuncta Walker (Lepidoptera: Geometridae) (Wong et al. 1985) and Neachrostia bipuncta Sugi (Lepidoptera: Noctuidae) sex pheromones (Ando et al. 1995). 6,7-epoxy-(Z,Z )- 3,9-octadecadiene has also been detected as a sex pheromone component in certain species. For instance, this compound is a major component of Erannis golda Djakonov (Lepidoptera: Geometridae) and Rivula leucanioides Walker (Lepidoptera: Noctuidae) sex pher- Fig. 7. Curve of the EAG dosage response of male E. obliqua to sex phero- omones (Ando et al. 1995). Polyunsaturated hydrocarbons with a mone compounds. C17–C23 straight chain and epoxy derivatives constitute the second major class of lepidopteran sex pheromones, which are referred to as type II pheromones (Ando et al. 2008). Approximately 100 lepi- 9-octadecatriene and 6,7-epoxy-(Z,Z )-3,9-octadecadiene attracted dopteran species release type II pheromones. The Noctuidae and the highest number of males, i.e., a mean of 45 males per night, with Geometridae families constitute a major class of type II sex phero- the maximum catch being 92 males in one night. During the field- mone (Ando et al. 2004). trapping experiment, no males were caught in the traps baited with (Z,Z,Z )-3,6,9-octadecatriene and 6,7-epoxy-(Z,Z )-3,9-octade- single components. cadiene both evoked significant EAG responses; nonetheless, the latter elicited an approximately twofold higher EAG response than the former did. Although epoxide is significantly less volatile than the Discussion corresponding triene hydrocarbon, less material is delivered per puff E. obliqua virgin females started calling at the start of the first scoto- to the antennae. This finding indicates that the antenna is particu- phase and continued calling throughout this phase, which suggests larly sensitive to epoxide. However, the wind-tunnel experiment that females are sexually mature at emergence. The mating initiation results demonstrated the importance of both triene and epoxide in rhythm can be controlled by artificial photoperiods; in this study, attracting male E. obliqua; no males were recovered from the this control was achieved by reversing the scotophase and photo- traps that contained triene or epoxide alone in the wind tunnel. phase with light. By contrast, a mixture of different ratios of triene and epoxide 174 Journal of Economic Entomology, 2016, Vol. 109, No. 1

Table 1. Behavioral response of male E. obliqua to synthetic compounds and female sex gland extract

Composition of the lure (lg) Behavioral response (%)a

(Z,Z,Z)-3,6,9-octadecatriene 6,7-epoxy-(Z,Z)-3,9-octadecadiene WF TF OR SC 10 0 0c0c0c0c 9 1 10c 10c 0c 0c 8 2 20c 20c 0c 0c 7 3 20c 20c 0c 0c 6 4 50b 50b 40b 10c 5 5 60b 60b 50b 20c 4 6 100a 100a 100a 90a 3 7 40b 20c 10c 0c 2 8 50b 50b 40b 10c 1 9 20c 20c 0c 0c 0 10 0c0c0c0c Female sex gland extract(10FE) 100a 100a 80a 60b

aWF, wing fanning; TF,taking ﬂight; OR, orientation; SC, source contact. N ¼ 20. Different letters indicate signiﬁcant difference at P < 0.05 according to Ryan’s multiple comparison test on proportions.

Table 2. Field trapping of male tea looper by using different blends

enantiomer forms; although our results showed that the racemic Downloaded from of components (2014) mixture exhibited biological activity, the enantiomeric configuration of the active pheromone component requires further elucidation. At Ratio of component Males caughta Most males (lg)(Z,Z,Z)-3,6,9-octadecatriene: (captured/night) caught by a present, we are attempting to synthesize optical isomers to deter- 6,7-epoxy-(Z,Z)-3,9-octadecadiene lure at a night mine the enantiomeric configuration or enantiomer rate of E. obliqua sex pheromones.

10:0 0 – http://jee.oxfordjournals.org/ 8:2 2 6 0.5c 6 6:4 6 6 2.9c 17 Acknowledgments 5:5 25 6 9.1b 56 4:6 45 6 8.3a 92 We are grateful to Jianghua Sun and Wei Wei for their help with the gas 2:8 18.5 6 7.6b 39 chromatography–electroantennogram analysis. We would also like to express 10:0 0 – our thanks to Dr. Henry Thompson of Colorado State University, CO, USA, Blank trap 0 – for his critical comments on the early draft. This research was supported by the National Natural Science Foundation of China (31200490 31170616 and aData in the table are presented as M 6 SE; the letters are the results of 31300426) and Anhui Major Demonstration Project for Leading Talent by guest on March 21, 2016 multiple comparison. Different letters indicate signiﬁcant difference at 0.05 Team on Tea Chemistry and Health. level. References Cited

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